The estrogen-response module (mERM) of the mouse lactoferrin gene which contains an overlapping COUP transcription factor (COUP-TF) and estrogen receptor (ER) binding element, is responsible for estrogen induction. Mutation and deletion of the COUP-TF binding element or reduction of the endogenous COUP-TF increase mERM estrogen-responsiveness. Likewise, overexpressing the COUP-TF expression vector blocked the estrogen- stimulated response of mERM in transfected cells. The molecular mechanism of this repression is due to the competition between COUP-TF and ER for binding at identical contact sites in the overlapping region of the mERM. There is no evidence of heterodimer formation between these two proteins. Our results indicate that two members of the steroid hormone-receptor superfamily work in concert to modulate lactoferrin gene expression. The 5'-flanking region of the human lactoferrin gene was also isolated from a human placenta genomic library. We sequenced 1294 bp of the 5'-flanking sequence, 70 bp of the first exon and 690 bp of the first intron. A major transcription start site was mapped by primer extension. The region immediately upstream from the transcription initiation site, following the first exon is abundant in G and C nucleotides. The promoter was comprised of a noncanonical TATA box (ATAAA), CAAT-like sequence (CAAC), and SP 1 binding site. A PU box, two half palindromic EREs (GGTCA), and an imperfect ERE overlapping with COUP-TF binding site were clustered within a 300 bp stretch (-465 to -165) of the DNA. The elements were also found in the corresponding positions in the mouse lactoferrin gene. Lactoferrin expression in human endometrium cells was stimulated by estrogen and the COUP-ERE element acted as an enhancer in response to the estrogen stimulation. In vitro DNase I footprinting analysis showed binding of the estrogen-receptor on the imperfect ERE. In contrast to the mouse lactoferrin COUP/ERE element, COUP-TF does not interact with this element. Therefore, the molecular mechanisms of the estrogen action that governs the lactoferrin gene expression are different between mouse and human.